近日，國(guó)際著名雜志Virology Journal在線(xiàn)刊登了中國(guó)科學(xué)院上海巴斯德研究所關(guān)于構(gòu)建手足口病相關(guān)的柯薩奇病毒A16型（coxsackievirus A16,，CVA16）感染性克隆的最新研究成果,。
手足口病是5歲以下兒童中發(fā)生的常見(jiàn)傳染病,，僅2010年和2011年，全國(guó)共有3,，394,，375例感染，其中死亡1414例,，對(duì)于兒童的健康成長(zhǎng)造成嚴(yán)重威脅,，但目前尚無(wú)針對(duì)手足口病的預(yù)防性疫苗或治療性藥物。
CVA16和人腸道病毒71型(enterovirus 71,，EV71)是引起手足口病的主要病原體,，因此,，了解CVA16和EV71基因組中各個(gè)區(qū)域的功能，對(duì)加強(qiáng)CVA16和EV71的病毒學(xué)和免疫學(xué)研究,，盡快研制出有效的預(yù)防和治療方法,，保障中國(guó)兒童的生命健康有非常重要的意義。然而,，EV71和CVA16均屬于RNA病毒,，直接對(duì)它們的基因組進(jìn)行遺傳操作非常困難。
上海巴斯德所研究生劉菲,、劉慶偉,、蔡一村等在黃忠研究員指導(dǎo)下，通過(guò)分子生物學(xué)手段,，提取了CVA16病毒的基因組RNA,，并且進(jìn)行體外反轉(zhuǎn)錄操作獲得與病毒基因組相對(duì)應(yīng)的cDNA序列，將此序列構(gòu)建到一個(gè)表達(dá)載體中,，獲得攜帶感染性cDNA克隆的質(zhì)粒,。利用該質(zhì)粒可以包裝出具有感染性的拯救病毒,。通過(guò)觀(guān)察細(xì)胞病變,、蛋白質(zhì)印跡以及免疫熒光等方法證實(shí)，拯救病毒與野生病毒的功能完全一致,。同時(shí),，測(cè)序結(jié)果還表明，拯救病毒的基因組具有穩(wěn)定的遺傳性,。
利用新構(gòu)建出的感染性克隆,，科研人員可以很方便地對(duì)RNA病毒的基因組進(jìn)行體外操作，從而對(duì)病毒的結(jié)構(gòu),、功能等多方面進(jìn)行研究,。更長(zhǎng)遠(yuǎn)地來(lái)說(shuō)，CVA16感染性克隆為今后闡明病毒的致病性,，開(kāi)發(fā)針對(duì)EV71/CVA16的預(yù)防性疫苗和治療性藥物等提供了研究基礎(chǔ),。
該研究得到中國(guó)科學(xué)院“百人計(jì)劃”、生化工程國(guó)家重點(diǎn)實(shí)驗(yàn)室開(kāi)放基金及賽諾菲-安萬(wàn)特-中科院上海生命科學(xué)研究院優(yōu)秀青年人才基金的資助,。（生物谷Bioon.com）
doi:10.1186/1743-422X-8-534
PMC:
PMID:
Construction and characterization of an infectious clone of coxsackievirus A16
Fei Liu†, Qingwei Liu†, Yicun Cai†, Qibin Leng and Zhong Huang*
Background Coxsackievirus A16 (CVA16) is a member of the Enterovirus genus of the Picornaviridae family and it is a major etiological agent of hand, foot, and mouth disease (HFMD), which is a common illness affecting children. CVA16 possesses a single-stranded positive-sense RNA genome containing approximately 7410 bases. Current understanding of the replication, structure and virulence determinants of CVA16 is very limited, partly due to difficulties in directly manipulating its RNA genome.
Results Two overlapping cDNA fragments were amplified by RT-PCR from the genome of the shzh05-1 strain of CVA16, encompassing the nucleotide regions 1-4392 and 4381-7410, respectively. These two fragments were then joined via a native XbaI site to yield a full-length cDNA. A T7 promoter and poly(A) tail were added to the 5' and 3' ends, respectively, forming a full CVA16 cDNA clone. Transfection of RD cells in vitro with RNA transcribed directly from the cDNA clone allowed the recovery of infectious virus in culture. The CVA16 virus recovered from these cultures was functionally and genetically identical to its parent strain.
Conclusions We report the first construction and characterization of an infectious cDNA clone of CVA16. The availability of this infectious clone will greatly enhance future virological investigations and vaccine development for CVA16
